Study on the hypolipidemic effect of Inonotus obliquus polysaccharide in hyperlipidemia rats based on the regulation of intestinal flora

Abstract The purpose of this study was to observe the effect of Inonotus obliquus polysaccharide (IOP) on blood lipids and its regulation on the intestinal flora in hyperlipidemia rats, and explore the modern biological connotation of IOP in reducing blood lipids. In this study, we obtained the crude IOP by the water extraction and alcohol precipitation method, and then classified it by DEAE ion‐exchange chromatography to obtain the acidic I. obliquus polysaccharide (IOP‐A). After the administration of the IOP‐A, the serum TC, TG, and LDL‐C levels were significantly lower, while the serum HDL‐C levels were significantly higher. The expression of CYP7A1 protein was considerably increased, whereas the expression of SREBP‐1C protein was considerably decreased in the rat hepatic tissue. In addition, the IOP‐A could significantly alleviate the hepatocyte fatty degeneration in the liver lobule of rats. We believe that the IOP‐A can affect the composition of intestinal flora by reducing the relative abundance of Firmicutes and increasing the relative abundance of Bacteroidetes. These findings indicated that the IOP‐A can regulate the dyslipidemia of hyperlipidemia rats, and its mechanism may be through regulating the CYP7A1 and SREBP‐1C expression in the metabolism of lipids, and correcting the imbalance of intestinal flora structure caused by a high‐fat diet.

Inonotus obliquus, also known as birch mushroom, chaga, Tricholoma obliquus and Ganoderma sibirica, belongs to Poria hypobrunnea Petch, Polyporaceae, Polyporales, Hymenomycetes, Basidiomycotina, Eumycota, and it parasitizes on the damaged parts of trunk or bark of birch, elm and red poplar, and is one of the world's top 10 precious medicinal fungi (Liu et al., 2015). I. obliquus contains more than 200 bioactive substances, such as terpenoids, steroids, corticosteroids, melanin, polyphenols, and polysaccharides (Jiang et al., 2017). I. obliquus polysaccharide (IOP) is the main active substance of I. obliquus, with the physiological functions of anti-cancer, anti-inflammation, antioxidation, hypoglycemia, and enhancing immunity (Ding et al., 2017;Xia et al., 2018). Previous work in our laboratory has proved that it has an obvious hypolipidemic effect, but its mechanism is not clear yet.
Intestinal flora can play a key role in the body's physiological regulation, such as the host's material metabolism to protect the host, and playing a role in the immunity, anti-tumor and improving the liver function of the host . Intestinal flora can determine the occurrence and progression of metabolic disorders by affecting the host's energy metabolism and immune system. The pathogenesis of hyperlipidemia may be closely related to intestinal flora since an imbalance of intestinal microecological flora is often found in hyperlipidemia patients, and the imbalance can conversely aggravate lipid metabolism disorder, leading to a vicious circle (Lv et al., 2018). It has been found that polysaccharides can improve the integrity of intestinal barrier function, reduce the intestinal mucosal damage, promote the growth and reproduction of beneficial bacteria related to intestinal hyperlipidemia, and inhibit the growth and reproduction of harmful bacteria to regulate and maintain their normal physiological activities by shaping the intestinal flora (Du et al., 2013;El Kaoutari et al., 2013;El-Hindawy, 2022). The products from polysaccharides degraded by intestinal flora, including lactic acid, acetic acid, propionic acid, and butyric acid, can regulate the pH and microbial diversity in intestines, and provide energy for the body, playing a key role in the protection of the normal peristalsis and barrier of intestines (El-Hindawy, 2022;Okeke et al., 2014;Uebanso et al., 2017). Further study on whether IOP can exert its hypolipidemic activity by regulating intestinal flora is needed.
The acidic I. obliquus polysaccharide (IOP-A) was extracted and separated, a high-fat diet was given to rats to establish a rat hyperlipidemia model for observing the hypolipidemic effect of the IOP-A, and the relationship between its hypolipidemic effect and the regulation of intestinal flora was investigated based on the regulation of intestinal flora to reveal the hypolipidemic mechanism in this study, which was expected to provide such a scientific foundation for the further implementation of functional foods and drugs related to I. obliquus.

| Materials
Specific Pathogen Free-grade male SD rats (4-5 weeks old and 190.0 ± 2.0 g) were provided by Changchun Yisi Experimental Animal Technology Co., Ltd. With animal license SCXK (Ji)-2016-0003. An ordinary feed (Rat pellet feed) and a high-fat feed were purchased from Jilin Medical University, and the formula of high-fat feed is shown in Table 1

| Preparation of the IOP-A
Inonotus obliquus was ground into powders at room temperature, and the I. obliquus powders were sifted through a 60-mesh sieve.
The sifted I. obliquus powders were dried to the constant weight after removing impurities with anhydrous ethanol, then immersed in deionized water overnight at a substance to liquid ratio of 1:20 (w/v), and extracted by heat reflux extraction method at 90°C for 2 h, which was repeated three times . The extracts were filtered and mixed, then the mixture was concentrated by vacuum decompression at 60°C, and the concentrated filtrate was added with four times the volume of anhydrous ethanol for the precipitation of the extract overnight.
Then, the precipitate was dissolved in distilled water and a dialysis bag with a molecular mass of 3500 Da was used to dialyze the precipitate-distilled water solution in distilled water for 48 h for removing the small molecular compounds in it, then lyophilized at −80°C for obtaining a crude IOP, and the crude IOP was classified by DEAE ion-exchange chromatography to obtain the IOP-A .

| Analysis of chemical composition and monosaccharide composition of IOP and IOP-A
Phenol-sulfuric acid, m-hydroxybiphenyl and Coomassie brilliant blue methods were used to determine the content of total polysaccharide, uronic acid and protein in IOP and IOP-A, in which glucose, D-galacturonic acid and bovine serum albumin were used as the standard substances, respectively.
Two mg of the IOP-A were dissolved in 1 ml of 2M HClmethanol solution, hydrolyzed at 80°C for 8 h, and then hydrolyzed for 1 h at 120°C with 1 ml of 2M trifluoroacetic acid.
1-phenyl-3-methyl-5-pyrazolone was used to precolumn-derivatize the hydrolysate, and a Shimadzu High-Performance Liquid Chromatography system (SPD-10AVD UV-VIS detector and LC-10ATvp pump) and DIKMA Inertsil ODS-3 (4.6 mm × 150 mm) were used to detect it. According to the retention time and peak area of standard monosaccharide, the monosaccharide composition was identified and the content was calculated (Onul et al., 2012).

| Establishment of rat hyperlipidemia model and rat administration
Male Sprague Dawley rats were divided into four groups randomly, namely the blank control group (NG), the model group (MG), the lovastatin group (PG), and the acidic I. obliquus polysaccharide group (IOP-A), eight rats in each group. Rats in the NG group received 100 g/kg of the basal diet daily and those in the MG, PG and IOP-A groups with 100 g/kg of the high-fat diet as described above daily. At the same time, 8.4 mg/kg of lovastatin was administered to rats in the PG group, 450 mg/kg of the IOP-A was administered to rats in the IOP-A group, and an equivalent amount of distilled water was administered to rats in the MG and NG groups once daily by gavage . The high-fat feeding and administration took place for an 8-week duration. All experiments using experimental animals were reviewed and approved by the Experimental Animal Committee of Beihua University.

| Measurement of body weights and organ indexes
During feeding, the body weights of rats were measured once weekly, the liver and spleen of rats were removed and washed with normal saline at the eighth week after the start of the feeding period . The liver index and spleen index were calculated according to the following equation after weighing the wet liver and spleen.

| Biochemical indicator determination
The rats were anesthetized with urethane, their blood was collected through the abdominal aorta. The blood was separated by centrifugation at 1917 g for 15 min to obtain serum, which was then stored at −20°C. The serum levels of HDL-C, LDL-C, TC and TG were determined following the kit manufacturers' instructions .

| Observation on the pathological changes of hepatic tissue
The hepatic tissue was fixed in 10% neutral formalin fix solution for the routine pathological sampling, sectioning and H&E staining . Pathological changes in the liver tissue were examined under an optical microscope (Dechesne et al., 2016). and TransGen AP221-02: TransStartFastpfu DNA polymerase reaction system was applied for the PCR amplification, in which each sample was replicated three times. The PCR products in the same sample were mixed, and the mixture was detected by 2% agarose gel electrophoresis. An AxyPrepDNA gel extraction kit was used to recover the PCR products after gel cutting, and then Tris-HCl was used to elute them and 2% agarose gel electrophoresis was conducted to detect them. The PCR products were quantified using a QuantiFluor™-ST blue fluorescence quantitative system according to the preliminary quantitative results of electrophoresis, and then mixed in the corresponding proportions. In accordance with the overlapping relationship, paired-end reads obtained by sequencing were firstly spliced, and the sequence quality was controlled and filtered. The Operational Taxonomic Units (OTU) diversity, alpha diversity, beta diversity, and comparison in biological taxonomy were performed for the analysis of the species differences among groups after distinguishing the samples.

| Western blotting analysis
An appropriate amount of RIPA protein lysis buffer was transferred into 100 mg of the hepatic tissue, and the solution was homogenized with a homogenizer. After 1 h of lysis on ice, the homogenate was centrifuged (4°C, 10 min) to obtain the supernatant . The BCA method was used to draw the standard curve, and the concentration of proteins was determined and adjusted. The SDS-PAGE electrophoresis on the proteins was performed to separate them, and then they were transferred onto PVDF membranes. Then, the membranes were blocked with 5% skimmed milk powder and shaken at room temperature for 2 h; after washing them, the membranes were incubated with the primary antibody diluents of CYP7A (1: 1:1000), SREBP-1C (1:1000), and β-actin (1:20000) overnight; after washing them, the membranes were incubated with the secondary anti-Rabbit antibody (1:2000) at room temperature for 1 h . After washing the membranes, the images were developed and photographed by a gel imager after the membranes were incubated with ECL luminescent liquid.

| Statistical analysis
The data from all the groups were measured three times. The data were presented as mean ± standard deviation (x ± s). The statistics analysis was performed using IBM SPSS Statistics for Windows, version 20.0 (IBM Corp.). The differences between the control group and the experimental group were evaluated by t-test. p < .05 means that there is a difference in the data, with a statistical significance.

| Extraction of IOP
The dialysis with a 1000 Da dialysate bag was performed to obtain IOP with a yield of 8.4% of the raw material, in which the ash was removed.
There were no significant differences in body weights between the four groups before the administration of the different agents.
The rats' body weights increased continuously in the NG group after the administration, while those in the MG group increased rapidly on the fourth week after the administration and increased significantly compared with those in the NG group on the ninth week (p < .05); the increasing trend in the PG group was comparable to that in the IOP-A group, whereas the body weights in the IOP-A group were less than those in the MG group, especially significantly on the ninth week (p < .05), indicating a slowly increased body weight in the IOP-A group, as shown in Figure 1.
As shown in Table 4, the liver index or the spleen index of rats in the MG group was considerably higher than that in the NG group, respectively (p < .05), while the liver index and the spleen index in the PG group and the IOP-A group were considerably lower than those in the MG group (p < .05).

| Effects of the IOP-A on biochemical indicators of rats
As seen in Table 5, compared with those in the NG group, the levels of TC, TG and LDL-C were considerably increased (p < .05), and those of HDL-C were considerably decreased (p < .05) in the serum of rats in the MG group; compared with those in the MG group, the levels of TC, TG and LDL-C were considerably decreased (p < .05), and those of HDL-C were considerably increased (p < .05) in the serum of rats in the PG group, and the levels of TC, TG and LDL-C were considerably decreased (p < .05), and those of HDL-C were considerably increased (p < .05) in the serum of rats in the IOP-A group.

| Effects of the IOP-A on pathological changes in the hepatic tissue of rats
No pathological abnormality in the hepatic tissue of rats was found in the NG group, with a normal hepatocyte size, an orderly arranged hepatic cord, and no fat vacuole and fat infiltration in hepatocytes ( Figure 2a). Some pathological changes could be found in the hepatic tissue of rats in the MG group, such as significantly increased steatosis hepatocytes, extremely swollen and edematous hepatocytes, more hepatocytes with a round shape and disordered hepatic cords ( Figure 2b). However, in contrast to MG group, fat vacuoles ( Figure 2d) were significantly reduced and hepatic cords were more closely arranged in the PG group ( Figure 2c) and the IOP-A group (Figure 2).

| Comparison of intestinal flora at OTU level in rats
Differences and similarities among the various samples were statistically analyzed at the OTU level, and the curve tended to be horizontal, suggesting that the sample size of this experiment was sufficient to ensure the accuracy of the results, as shown in Figure 3a. 1791 OTUs were shared in all the samples, and the results showed a species abundance of the NG group > the IOP-A group > the MG group, as shown in Figure 3b.

| Alpha diversity analysis of intestinal flora in rats
The Chao1 index and observed_species indicated the NG group > the IOP-A group > the MG group in the number of species, and the Simpson and Shannon indexes both indicated that the richness and evenness of the IOP-A group were higher than those in the MG group, while the diversity of the total bacterial community in the NG group was higher, as shown in Figure 4.

| Beta diversity analysis of intestinal flora in rats
The abscissa indicates the first principal component, and the contri-   Figure 6).

| Comparison of the intestinal flora in rats at the genus level
A total of 20 bacterial communities were identified at the genus level in the rectal fecal microorganisms of the NG, MG, and IOP-A groups (Figure 7), including 20 bacterial communities, considerably up-regulated (p < .05) and that of Streptococcus was considerably down-regulated (p < .05) in the IOP-A group (Figure 7).

| Effects of the IOP-A on CYP7A1 and SREBP-1C protein expressions in the hepatic tissue of rats
The expression of SREBP-1C protein in the MG group was considerably higher than that in the NG group (p < .05), while the expression of CYP7A1 protein in the MG group were considerably lower than that in the NG group (p < .05); the expression of SREBP-1C protein in the PG and IOP-A groups were considerably lower than that in the MG group (p < .05), while the expression of CYP7A1 protein in the PG and IOP-A groups were considerably higher than that in the MG group (p < .05; Figure 8).

| DISCUSS ION
Hyperlipidemia is a metabolic disorder syndrome characterized by abnormal increase of lipid constitutes in plasma, and has been recognized as one of the main causes of fatty liver now (Chang et al., 2015). The liver is considered to be a vital organ for the uptake of fat, the metabolism of free fatty acid, and synthesis and secretion of cholesterol, phospholipid and lipoproteins, and when the fat in the blood exceeds the metabolic capacity of the liver, fat will be deposited in hepatocytes, leading to the degeneration of hepatocytes, and eventually fatty liver (van Driel et al., 2016;Zhang et al., 2016).
Hepatomegaly can be induced by the intake of a high-fat diet for long term, and an increased liver index and an increased spleen index may indicate hyperlipidemia to a certain extent (Cho et al., 2017;Junior et al., 2016). A rat hyperlipidemia model was induced by a high-fat diet in this study, and the liver index and spleen index increased significantly, the number of steatosis hepatocytes increased significantly and the arrangement of hepatic cords were disordered, and of TC, TG and LDL-C levels in the serum increased significantly, while the HDL-C level decreased significantly in the hyperlipidemia rats . After the administration of the IOP-A, the liver index and spleen index were decreased, the fatty liver was relieved, the serum TC, TG and LDL-C levels were significantly lower, while the serum HDL-C level was significantly higher, indicating that the IOP-A can effectively regulate lipid metabolism in hyperlipidemia rats. Intestinal microflora is considered to be involved in lipid metabolism, and the intestinal absorption is the basis for Chinese medicines to exert their effects, so intestinal flora is likely to be a new target for regulating lipid metabolism disorders (Fadrosh et al., 2014). The high-fat diet-induced hyperlipidemia will destroy the balance of intestinal flora, leading to lipid metabolism disorders, and then more serious hyperlipidemia, which may form a vicious cycle . It was found in our study that there were differences in the number of OTUs among the groups, and the analysis of alpha diversity and beta diversity showed that there were differences in the richness of intestinal flora, that is, the lower species richness in the MG group and the significantly higher spe-  (Bortolin et al., 2018;Yin et al., 2015). As reported, fungal polysaccharides inhibit Firmicutes to a large extent, thereby tending to balance Bacteroidetes, improve the expression of dominant bacteria in the intestinal tract of rats, and regulate the composition of the intestinal flora . Our study revealed that the number of Firmicutes and Bacteroidetes was higher in the NG, MG, and IOP-A groups.
Following the administration of the IOP-A to hyperlipidemia rats, the relative abundance of Firmicutes decreased, and the propor- Bile acids produced by gut microbes affect hyperlipidemia (Duan et al., 2021). Bile acids, the major metabolites of cholesterol in the liver, are formed by the CYP7A1 enzyme and enhance the absorption of fats, nutrients, and lipophilic vitamins as well as regulate lipid, glucose, and energy metabolism. Aside from that, gut microbes are capable of degrading carbohydrates into monosaccharides and converting them into hydrogen, carbon dioxide, methane, and short-chain fatty acids, which provide energy for the host (Mahamuni et al., 2012). As a key transcription factor in the metabolism of TG, SREBP-1C can regulate the synthesis of fat and fatty acids following excessive carbohydrate intake (Tang et al., 2017). The study found that after administration of the IOP-A to the MG group, the expression of CYP7A1 protein in the hepatic tissue was considerably increased, and the expression of SREBP-1C protein in the hepatic tissue was considerably decreased. And the relative abundance of Firmicutes and the ratio of Firmicutes to Bacteroidetes decreased in the intestinal microflora.
Consequently, the IOP-A could exert hypolipidemic effects by activating the CYP7A1 enzyme involved in cholesterol metabolism, inhibiting the SREBP-1C factor in lipid metabolism and regulating the balance of the intestinal microflora structure.
In this study, the hypolipidemic activity of the IOP-A and its correlation with the regulation of intestinal microflora, and the target of the IOP-A at the molecular level, were clarified, which may lay a foundation for the study on the action of I. obliquus and its mechanism.

| CON CLUS ION
The IOP-A has a hypolipidemic effect, which may be due to its ability to regulate the expression of CYP7A1 and SREBP-1c, as well as to correct an imbalance in the structure of the intestinal microflora. The study clarifies that IOP-A regulates the expression of CYP7A1 and SREBP-1c, and explores part of the mechanism, but it cannot rule out the combined effects of other pathways on blood lipid regulation. The composition of I. obliquus is complex, and its anti-hyperlipidemic mechanism needs further comprehensive and in-depth research. Consequently, the experiment provides experimental and theoretical support for the further development of functional foods and medicines based on I. obliquus.

ACK N OWLED G M ENTS
Thanks for the financial support from the National Natural Science

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

R E FE R E N C E S F I G U R E 8 Effects of the IOP-A on CYP7A1 and SREBP-1C
protein expressions in the hepatic tissue of rats. (a) Relative expressions of SREBP-1C and CYP7A1 protein in the hepatic tissue of rats; (b) Electrophoretic map of SREBP-1C and CYP7A1 expressions in the hepatic tissue of rats. n = 8; Compared with the NG group, *p < .05; compared with the MG group, # p < .05. IOP-A, acidic Inonotus obliquus polysaccharide group; MG, the model group; NG, the normal group; PG, positive group